Abstract: Craniofacial malformations, including cleft palate and craniofacial skeletal defects, are among the most common structural birth defects in humans. Using a phenotype-driven genetic screen to identify new genes that play important roles in craniofacial development, we isolated an N-ethyl-N-nitrosourea (ENU)-induced mutation that causes cleft palate and skeletal dysplasia in mice. The specific mutant phenotypes resemble that previously described in Fgf18-/- mutant mice, including cleft palate and shorter limbs with angulated radius and bowed tibia. However, targeted sequencing of the Fgf18 gene did not find any mutation. Using whole exome sequencing followed by allele-specific genotyping, the cleft palate and skeletal defects in this mutant mouse line have been found to completely co-segregate with homozygosity of a nonsense mutation in the Adamts6 gene, Adamts6Q999*. Remarkably, mice homozygous for an independent ENU-induced missense mutation, Adamts6S149R, exhibit similar cleft palate and limb defects. Whereas little is currently known about the cellular and molecular functions of ADAMTS6, the primary sequence of the ADAMTS6 protein identifies it as a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin motifs) family of secreted, extracellular matrix (ECM)-associated metalloproteinases. Structurally, ADAMTS6 is most closely related to ADAMTS10, which has been shown to bind to fibrillin-1 (FBN1), a major component of the ECM fibrils. FBN1 has been implicated in regulating TGF? signaling by sequestering the TGF? ligands in latent complexes in the ECM. Since TGF? signaling plays crucial roles in craniofacial and skeletal development and since FGF signaling is also modulated by interactions between the FGF ligands and ECM components, we hypothesize that ADAMTS6 regulates craniofacial and skeletal development through regulation of TGF? and/or FGF18 signaling. We propose two specific aims for this Exploratory/Developmental Grant application: (1) to test the hypothesis that ADAMTS6 regulates FGF18 and/or TGF? signaling during craniofacial and skeletal development, and (2) to generate Adamts6 conditional mice using CRISPR/Cas9-mediated genome editing and to elucidate cell type-specific ADAMTS6 functions in craniofacial and skeletal development. These studies explore novel aspects of the molecular and cellular mechanisms of craniofacial development and will significantly advance the understanding of ADAMTS function in development and diseases.